Aging is a major contributor to cerebrovascular disease and subsequent neurological sequelae. The frequency and severity of these diseases, age of onset, and underlying mechanisms differ between men and women and are augmented by age-related diseases such as type 2 diabetes (T2D). The endothelium, a principle component of the microcirculation, is particularly sensitive to the negative effects of aging or T2D and mitochondria appear to play a pivotal role. Progress in elucidating the underlying mechanisms negatively affecting endothelial mitochondria and developing beneficial therapies has been obstructed due to the inability to follow mitochondrial characteristics in the brain microcirculation in real time during the development of aging and age-associated diseases. To address this deficiency, we will use three new approaches. First, we will use a mouse model which we developed with genetic labeling of mitochondria only in endothelium with Dendra2 fluorescent protein (mitoDendr2 FP). Second, we will use a novel, high throughput method we developed that allows the determination of energy production by mitochondrial respiration and glycolysis in freshly harvested brain microvessel from the mouse. Glycolysis is a major energy producing process in the endothelium and the relative importance of oxidative phosphorylation (OXPHOS) and glycolysis changes with aging and T2D is unclear. We also will examine whether mitochondrial fuels change. Third, we will use RNA Sequencing and Proteomics to explore underlying mechanisms involving energy producing pathways. Our preliminary and published data have led to the overall hypothesis that mitochondria play key roles in adverse changes in the cerebral microcirculation during aging and T2D and that therapies targeting mitochondria are protective. Thus, we speculate that mitochondria in microvessels are adversely affected more and earlier than large arteries during aging, T2D acerbates changes in mitochondria in microvessels, glycolysis becomes a more important source of ATP during aging and T2D, alternative fuel sources for OXPHOS become important during aging and T2D, mitochondria are more resilient in female microvessels, and mitochondria represent a useful therapeutic target to protect microvessels. We have 2 aims. Aim 1: Elucidate mechanisms of mitochondrial and vascular changes during aging. We will: a) determine mitochondrial and vascular characteristics in vivo in male and female aged mice, b) determine effects of aging on glycolysis and OXPHOS, mitochondrial fuel, and mitoROS, c) elucidate mechanisms affecting mitochondrial and glycolytic dynamics, and d) explore treatment modalities. Aim 2: Determine mechanisms of mitochondrial and vasculature changes during aging and T2D. We will: a) determine mitochondrial and vascular characteristics in aged male and female mice during T2D, b) determine effects of aging on glycolysis and OXPHOS, mitochondrial fuel, and mitoROS, c) elucidate mechanisms involved in changes in mitochondrial and vascular dynamics, and d) explore therapeutic approaches to improve mitochondrial and vascular function.